The overall objective is to define the Ca2+-signaling pathways in macrophages that regulate functions of and genes involved in the inflammatory response. Obesity, Type 2 Diabetes and other disease states associated with the Metabolic Syndrome are inflammatory diseases characterized by infiltration of macrophages into metabolic tissue. We identified a CaM kinase cascade comprised of CaMKK2 and AMPK that regulates energy balance. Mice null for CaMKK2 are protected from the insulin resistance, glucose intolerance, obesity and liver steatosis that result from consuming a high fat diet. At least part of the protection is mediated via the central nervous system as CaMKK2 functions in neurons of the hypothalamic arcuate nucleus to control production of neuropeptide Y. However, the attenuated inflammatory response in the adipose tissues of obese CaMKK2 null mice led us to question whether there may also be a peripheral immune target for CaMKK2 and we found CaMKK2 is expressed in macrophages. Depletion of CaMKK2 from macrophages results in markedly attenuated response to the bacterial endotoxin, Lipopolysaccharide (LPS) that binds to and activates Toll-like receptor 4 (TLR4) and causes systemic inflammation. The altered macrophage responses include decreased inflammatory cytokine and chemokine gene expression and secretion, cell surface expression of co- stimulatory molecules, ability to phagocytose bacterial particles and motility. Accordingly, administration of a dose of LPS alone or in combination with N-galactosamine to CaMKK2 null mice that is lethal for WT mice results in a profound decrease in proinflammatory cytokines and chemokines in serum, reduced liver damage and well as markedly prolonged survival. We hypothesize that CaMKK2 plays a key role in mediating the cellular signals initiated by Toll-like receptors (TLRs), thereby affecting macrophage activation and inflammatory responses. To evaluate this hypothesis we will: 1) define the role of CaMKK2 in the activation of isolated macrophages by; (a) investigating the role of CaMKK2 in mechanisms regulating activation and de- activation of macrophages; (b) identifying targets of CaMKK2 critical for the responses to TLR4 agonists; (c) identify genes and transcription factors regulated by CaMKK2; and 2) define the role of CaMKK2 in the inflammatory response induced by over-nutrition by using conditional LysM-Cre/CaMKK2lox/lox, and conditional CaMKK2 Cre/lox mice already developed by our laboratory to; (a) determine if ablation of CaMKK2 in macrophages prevents inflammation of metabolic tissues and results in insulin sensitivity and glucose tolerance; (b) determine if the ablation of CaMKK2 reverses inflammation in metabolic tissues and restores insulin sensitivity and glucose tolerance. Because inflammation is such a prevalent cause of or response to numerous human diseases we believe our studies will reveal CaMKK2 to be an important, novel target for therapeutic intervention. PUBLIC HEALTH RELEVANCE: We have uncovered a novel function for a calcium-activated protein kinase, CaMKK2, in mechanisms regulating the response of macrophages to stimuli that lead to inflammation. As macrophages play a pivotal role in the regulation of the inflammatory response, and in turn, in the pathogenesis of clinically relevant conditions associated with an inappropriate inflammatory response such as obesity, Type 2 Diabetes, and atherosclerosis, CaMKK2 may be a novel target for treatment of these immune disorders.